Antisense oligonucleotides having tumorigenicity-inhibiting activity

ABSTRACT

The invention encompasses tumorigenicity-inhibitiig antisense oligonucleotide sequences complementary to mRNA or double-stranded DNA that encodes mammalian DNA methyl transferase. It further encompasses methods for inhibiting tumorigenicity and pharmaceutical composition comprising the tumorigenicity-inhibiting antisense nucleotide.

This is a divisional of application Ser. No. 08/161,673, filed Dec. 1,1993, U.S. Pat. No. 5,578,716.

FIELD OF THE INVENTION

This invention relates to oligonucleotides for use in the inhibition ofDNA methyl transferase expression, and more particularly, antisenseinhibition of tumorigenicity.

DESCRIPTION OF RELATED ART

Alterations in the normal gene expression profile of a cell are thoughtto be early events in oncogenic transformation. A large number ofoncogenes are transcription factors. However, many oncogenes are nottranscription factors but are involved in signal transduction pathwaysthat trigger activation of transcription factors such as the activationof Jun by the RAS signalling pathway.

The DNA methyltransferase (DNA MeTase) gene 5' region has recently beencharacterized (Rouleau et al, J. Biol. Chem., 267: 7368-7377 (1992)). Itcontains at least two functional AP-1 sites and the promoter of thatgene can be dramatically transactivated by Fos, Jun or Ras. The DNAMeTase gene encodes an activity that is responsible for methylatingcytosine residues in the dinucleotide sequence CpG. A hallmark of DNAmethylation is that 80% of the CpG sites are methylated in a nonrandommanner generating a site-, tissue- and gene-specific pattern ofmethylation. Methylation patterns are formed during development.Establishment and maintenance (Razin and Szyf, Biochim. Biophys. Acta,782: 331-342 (1984)) of the appropriate pattern of methylation iscritical for development (Li et al., Cell, 69: 915-926 (1992)) and fordefining the differentiated state of a cell (Szyf, et al., J. Biol.Chem., 267: 12831-12836 (1992)). The pattern of methylation ismaintained by DNA MeTase at the time of replication (Szyf et al., J.Biol Chem., 260: 8653-8656 (1985)); the level of DNA MeTase activity andgene expression is regulated with the growth state of different primaryand immortal cell lines (Szyf et al., J. Biol. Chem., 266: 10027-10030(1991)).

The relationship of DNA methylation to tumorigenicity has been in astate of confusion for some time. Some reports have suggested thathypomethylation of certain genes may be implicated in neoplasia (seee.g., Ohtani-Fukita et al., Oncogene, 8: 1063-1967 (1993)). However manyreports have demonstrated hypomethylation of total genomic DNA (seee.g., Feinberg et al., Cancer Res., 48: 1159-1161 (1988); Goelz andVogelstein, Science, 228: 187-190 (1985)). Still other reports haveconnected hypomethylation of individual genes with tumorigenicity (seee.g., Feinberg and Vogelstein, Nature, 301: 89-92 (1983); Jones andBuckley, Adv. Can. Res., 54: 1-12 (1990)). Moreover, it has beensuggested that current hypotheses about DNA methylation and cancersuggest that agents that reduce DNA methylation should causetransformation of cells (Jones and Buckley, supra). Thus, the prior artis devoid of any meaningful suggestion of how regulation of DNAmethylation may be successfully manipulated to diminish tumorigenicity.

Antisense oligonucleotide technology has allowed for inhibition ofexpression of a variety of genes. See generally Agrawal, Trends inBiotech., 10: 152 (1992). By binding to the complementary nucleic acidsequence In RNA, antisense oligonucleotides are able to inhibit splicingand translation of RNA. In this way, antisense oligonucleotides are ableto inhibit protein expression. Antisense oligonucleotides have also beenshown to bind to genomic DNA, forming a triplex, and inhibittranscription. Furthermore, a 17-mer base sequence statistically occursonly once in the human genome, and thus extremely precise targeting ofspecific sequences is possible with such antisense oligonucleotides.

In 1978 Zamecnik and Stephenson were the first to propose the use ofsynthetic antisense oligonucleotides for therapeutic purposes.Stephenson and Zamecnik, Proc. Natl. Acad. Sci. U.S.A., 75: 285 (1978);Zamecnik and Stephenson, Proc. Natl. Acad. Sci. U.S.A., 75: 280 (1978).They reported that the use of a oligonucleotide 13-mer complementary tothe RNA of Rous sarcoma virus inhibited the growth of the virus in cellculture. Since then, numerous other studies have been publishedmanifesting the in vitro efficacy of antisense oligonucleotideinhibition of viral growth, e.g., vesicular stomatitis viruses (Leonettiet al., Gene, 72: 323 (1988)), herpes simplex viruses (Smith et al,Proc. Natl. Acad. Sci. U.S.A., 83: 2787 (1986)), and influenza virus(Zerial et al., Nucleic Acids Res., 15: 9909 (1987)).

Antisense oligonucleotides have also been shown to inhibit proteinexpression from endogenous mammalian genes. For example, Burch andMahan, J. Clin. Invest., 88: 1190 (1991), disclosed antisenseoligonucleotides targeted to murine and human IL-1 receptors thatinhibited IL-1-stimulated PGE₂ synthesis in murine and humanfibroblasts, respectively; Colige et al., Biochemistry, 32: 7 (1993)disclosed antisense oligonucleotides that specifically inhibitedexpression of a mutated human procollagen gene in transfected mouse 3T3cells without inhibiting expression of an endogenous gene for the sameprotein; and Monia et al., J. Biol. Chem., 267: 19954 (1992), disclosedselective inhibition of mutant Ha-ras mRNA expression withphosphorothioate antisense oligonucleotide.

Although antisense approaches have shown promise for a variety ofdisease states, there is no clear message about how or whether anygenetic target exist that would allow successful use of antisenseapproaches to affect tumorigenicity. There is, therefore, a need todevelop this promising technology in a way that might allow it to beapplied in the fight against neoplasia.

SUMMARY OF THE INVENTION

Previous teachings have suggested that agents that inhibit DNAmethylation should be capable of transforming cells (see e.g., Jones &Buckley, Adv. in Cancer Res., 54: 1-23 (1990)).

The present invention provides antisense oligonucleotides thatsurprisingly demonstrate tumorigenicity-inhibiting activity. Theinventive oligonucleotides inhibit tumorigenisis by inhibitingexpression of the gene encoding DNA methyl transferase. Theseoligonucleotides are complementary to mRNA or double-stranded DNA thatencodes mammalian DNA methyl transferase. The present invention furtherprovides useful compounds, compositions and methods for preventing theexpression of the DNA methyl transferase gene. A still further object ofthe present invention is to provide compounds, compositions and methodsfor the treatment of and inhibition of tumorigenicity.

Accordingly, this disclosure presents antisense oligonucleotides thathave been constructed and are targeted to bind to nucleic acid sequencesencoding DNA MeTase, thereby blocking production of the expressionproduct. Also presented are methods for inhibiting DNA MeTase expressionand tumorigenesis.

The invention is useful in curing experimental mice of tumors. Morespecifically, the invention is useful in curing nude mice of humantumors, and, in particular, human small lung cell carcinoma. Theinvention may thus be used to avoid sacrificing an animal at the end ofan experiment.

The present invention provides methods for inhibiting tumorigenesis byexpressing an antisense message to the DNA MeTase in a cell line, andspecifically in mouse and human cancer cell lines. Expression of anantisense DNA MeTase leads to: (i) a general reduction in themethylation content of the genome; (ii) demethylation of regionsaberrantly methylated in a cell line such as the adrenal specific21-hydroxylase gene as well as tumor suppressor loci; (iii)morphological changes indicative of inhibition of the transformedphenotype; (iv) inhibition of tumorigenesis in vitro as well as a lossof angiogenic function; and (vi) to the ability to undergo an apoptoticdeath program under appropriate conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a physical map of the plasmids pZEM and pZαM. Themetallothionine (MT) promoter (shaded box), the human growth hormone 3'region (HGH) (open bar), and the MeTase cDNA sequences (hatched) areindicated.

FIG. 2 is a graph showing the state of methylation of total genomic DNAand specific genes in Y1_(p) ZαM transfectants. The spots on TLC platescorresponding to C and 5-methyl C were scraped and counted in a liquid βscintillation counter. The values represent the means ± SEM.

FIG. 3 is a graph indicating anchorage independent growth assay of: Y1pZEM (clones 4 and 7) and Y1 pZαM transfectants (clones 4, 7 and 9).

FIG. 4 is a graph indicating a loss of antisense expression in tumorsderived from Y1 pZαM transfectants.

FIG. 5a is a graph showing survival and apoptosis of Y1 pZαM cells asdetermined by a density restricted growth assay.

FIG. 5b is a graph showing survival and apoptosis of Y1 pZαM cells inserum deprived medium.

FIG. 6 is a graph showing the percentage of CpG methylation in NCI H446cells expressing antisense to DNA MeTase and in cells expressing a DNAMeTase sense control oligonucleotide.

FIG. 7 shown the ability of NCI H446 cells treated with antisense andcontrol oligonucleotides to grow in an anchorage independent fashion insoft agar.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention provides antisense oligonucleotides that surprisinglyinhibit tumorigenicity. These oligonucleotides are complementary to mRNAor double-stranded DNA that express mammalian, and, in particular, humanor mouse, DNA methyl transferase and unexpectedly displaytumorigenicity-inhibiting activity. Ones preferred antisenseoligonucleotide of the present: invention is 5'-CATCTGCCATTCCCACTCTA-3'(SEQ ID NO 1), having either phosphodiester or phosphorothioatelinkages. Other suitable antisense oligonucleotides include thephosphorothioate: 5'-TTGGCATCTGCCATTCCCACTCTA-3' (SEQ ID NO 2).

Modified oligonucleotides having in vivo activity against tumorigenicityare referred to herein as anti-tumorigenicity ortumorigenicity-inhibiting modified oligonucleotides. The inventionprovides tumorigenicity-inhibiting modified oligonucleotides that haveefficacy in inhibiting expression of DNA methyl transferase. Modifiedoligonucleotides according to the invention have specific preferredcharacteristics that are discussed in greater detail for each preferredembodiment below. In addition to these characteristics, modifiedoligonucleotides according to the invention may optionally haveadditional ribonucleotide, 2'-substituted ribonucleotide, and/ordeoxyribonucleotide monomers, any of which are connected together via 5'to 3' linkages which may include any of the internucleotide linkagesknown in the art. Preferably, such modified oligonucleotides mayoptionally contain phosphodiester, phosphotriester, phosphoramidate,siloxane, carbonate, carboxymethylester, acetamidate, carbamate,thioether, bridged phosphoramidate, bridged methylene phosphonate,bridged phosphoramidate, bridged methylene phosphonate, bridgedphosphorothioate and/or sulfone internucleotide linkages. Those skilledin the art will recognize that the synthesis of oligonucleotidescontaining any of these internucleotide linkages is well known to thoseskilled in the art, as is illustrated by articles by Uhlmann and-Peyman,Chemical Reviews, 90: 543-584 (1990) and Schneider and Banner,Tetrahedron Lett., 21: 335 (1990) Preferably, modified oligonucleotidesaccording to the invention should contain from about 6 to about 100monomers in total and most preferably from about 12 to about 50 totalmonomers. Such modified oligonucleotides may also optionally containmodified nucleic acid bases and/or sugars, as well as addedsubstituents, such as diamines, cholesteryl or other lipophilic groups.

Various preferred embodiments of modified oligonucleotides according tothe invention are discussed below. Although these embodiments all have anucleotide sequence from the same region of the DNA MeTase gene, thoseskilled in the art will recognize that the tumorigenicity-inhibitingefficacy of oligonucleotides having nucleotide sequences complementaryto other essential nucleic acid sequences of DNA MeTase can also beenhanced by incorporating into such oligonucleotides the structuralfeatures of preferred embodiments of modified oligonucleotides accordingto the invention.

For purposes of the invention, complementary means having a sequencethat hybridizes to the essential nucleic acid sequence underphysiological conditions. An essential nucleic acid sequence of the DNAMeTase gene moans a nucleic acid sequence that is required forexpressing DNA MeTase. For example, such oligonucleotides can have othersequences from the DNA MeTase gene. Indeed, any sequence from the DNAMeTase gone (the 5'-region as disclosed by Rouleau et al, J. Biol.Chem., 267: 7368-7377 (1992) or Yen et al., Nucl. Acids Res., 9:2287-2291 (1992) should serve as the basis for modified oligonucleotidesaccording to the invention. As a practical matter, the structuralfeatures of preferred embodiments of modified oligonucleotides accordingto the invention should enhance the tumorigenicity-inhibiting activityof any antisense oligonucleotide having a nucleotide sequence thathybridizes in a cell with any essential nucleic acid sequence of the DNAMeTase gene.

Each preferred embodiment of modified oligonucleotides according to theinvention is separately discussed in greater detail below.

In a first preferred embodiment, tumorigenicity-inhibiting modifiedoligonucleotides according to the invention are in the form of a mixedbackbone or chimeric oligonucleotide having one or more regions ofnucleotides connected by phosphorothioate or phosphorodithioateinternucleotide linkages ("phosphorothioate or phosphorodithioateregion") as well as one or more regions of nucleotides connected byalkylphosphonate internucleotide linkages ("alkylphosphonate region").In this embodiment, at least one alkylphosphonate region preferablyincludes nucleotides at or near the 5' end and/or the 3' end of theoligonucleotide. For purposes of the invention, "at or near" the 5' orthe 3' end of the oligonucleotide means involving at least onenucleotide within about 5 nucleotides from the 5' or 3' end of theoligonucleotide. Preferably, the alkylphosphonate region comprises fromabout 2 to about 10 contiguous nucleotides connected by alkylphosphonatelinkages. Preferably, the phosphorothioate or phosphorodithioate regioncomprises at least 3, and up to about 100 contiguous nucleotidesconnected by phosphorothioate or phosphorodithioate linkages. Manyembodiments of oligonucleotides having this type of backbone structureare taught in U.S. Pat. Nos. 5,149,797 and 5,220,007, the teachings ofwhich are hereby incorporated by reference.

Modified oligonucleotides having tumorigenicity-inhibiting activityaccording to this embodiment of the invention are synthesized by solidphase methods, alternating H-phosphonate chemistry and sulfur oxidationfor phosphorothioate regions, and alkylphosphonamidate chemistry foralkylphosphonate regions. A preferred H-phosphonate approach is taughtby Agrawal et al., U.S. Pat. No. 5,149,798, the teachings of which arehereby incorporated by reference. Alkylphosphonamidite chemistry is wellknown in the art, as illustrated by Agrawal and Goodchild, TetrahedronLett., 28: 3539-3542 (1987). synthesis of phosphorodithioate-containingoligonucleotides is also well known in the art, as illustrated by U.S.Pat. No. 5,151,510, the teachings of which are hereby incorporated byreference (See also, e.g., Marshall and Caruthers, Science, 259:1564-1570 (1993) and references cited therein).

In a second preferred embodiment, modified oligonucleotides havingtumorigenicity-inhibiting activity according to the invention are in theform of a mixed backbone of chimeric oligonucleotide having one or moreregion of nucleotides connected by phosphorothioate orphosphorodithioate internucleotide linkages ("phosphorothioate orphosphorodithioate region"), as well as one or more region ofnucleotides connected by alkylphosphonothioate or arylphosphonothioateinternucleotide linkages ("alkylphosphonothioate region"). In thisembodiment, at least one alkylphosphonothioate region preferablyincludes nucleotides at or near the 5' end and/or the 3' end of theoligonucleotide. Preferably, the alkylphosphonothioate region comprisesfrom about 2 to about 10 contiguous nucleotides connected byalkylphosphonothioate linkages. Preferably, the phosphorothioate orphosphorodithioate region comprises at least 3, and up to about 100contiguous nucleotides connected by phosphorothioate orphosphorodithioate linkages.

Tumorigenicity-inhibiting modified oligonucleotides according to thisembodiment of the invention are synthesized by solid phase methods,alternating chemistries for each region to be synthesized.Phosphorothioate or phosphorodithioate regions are synthesized asdescribed for the first embodiment. Alkylphosphonothioate regions aresynthesized by coupling together two or more nucleosides viaalkylphosphite linkages, then oxidatively thiolating the alkylphosphitelinkages to produce alkylphosphonothioate linkages (see e.g., Agrawal etal., Nucl. Acids Res., 20: 2729-2735 (1993).

In a third preferred embodiment, tumorigenicity-inhibiting modifiedoligonucleotides according to the invention are in the form of a hybridoligonucleotide having regions of deoxyribonucleotides("deoxyribonucleotide regions") and regions of ribonucleotides or2'-substituted ribonucleotides ("ribonucleotide regions"). Preferably,from about ones to about all of the internucleotide linkages arephosphorothioate or phosphorodithioate linkages. Preferred2'-substituted ribonucleotides are halo, amino, alkyl, aryl or loweralkyl (1-6 carbon atoms) substituted ribonucleotides, especially2'-OMe-ribonucleotides. Preferably, at least some of the ribonucleotideregions include nucleotides present at or near the 5' end and/or the 3'end of the oligonucleotide. Most preferably, the ribonucleaotide regionseach comprise from about 2 and preferably from about 4 to about 100contiguous ribonucleotides and/or 2'-substitute oligonucleotides Thedeoxyribonucleotide regions are optional, and when present may containfrom about 1 to about 100 contiguous deoxyribonucleotides.Tumorigenicity-inhibiting modified oligonucleotides according to thisembodiment of the invention are typically synthesized by solid phasemethods, preferably by the phosphoramidite approach, usingdeoxynucleotide phosphoramidites for deoxyribonucleotide regions, andribonucleotide or 2'-substituted ribonucleotide phosphoramidite forribonucleotide regions.

In a fourth preferred embodiment, tumorigenicity-inhibiting modifiedoligonucleotides according to that invention are in the form of anoligonucleotide having at its 5' and/or 3' end a cap structure thatconfers exonuclease resistance to the oligonucleotide. Such modifiedoligonucleotides preferably also have from 1 to about all modified(non-phosphodiester) internucleotide linkages. Preferred cap structuresinclude lower alkyl (C₁ -C₁₂) or alcohol groups. Preferred modifiedinternucleotide linkages include phosphotriester, phosphoramidate,siloxane, carbonate, carboxymethylester, acetamidate, carbamate,'thioether, bridged phosphoramidate, bridged methylene phosphonate,bridged phosphorothioate, sulfone, phosphorothioate andphosphorodithioate linkages. Tumorigenicity-inhibiting modifiedoligonucleotides according to this embodiment of the invention aresynthesized according to procedures well known in the art (see e.g.,Uhlmann and Peyman, Chemical Reviews, 90: 43-584 (1990); Schneider andBanner, Tetrahedron Lett., 31: 335 (1990)). For oligonucleotides havingcap structures at the 3' end, the cap structure is reversibly attachedto the solid-support and is then coupled to the first nucleotide monomerin the synthesis scheme. For oligonucleotides having cap structures atthe 5' end, the cap structure is coupled to the end of theoligonucleotide after addition of the last nucleotide monomer in thesynthesis scheme.

In a fifth embodiment, tumorigenicity-inhibiting modifiedoligonucleotides are self-stabilized by having a self-complementaryregion that hybridizes intramolecularly with the oligonucleotide to forman exonuclease resistant hairpin-like structure (see e.g., Agrawal etal., Nucleic Acids Res. 20: 2729-2735 (1993). Modified oligonucleotidesaccording to this embodiment of the invention are generallycharacterized by having two regions: a DNA MeTase hybridizing region anda self-complementary region. The DNA MeTase hybridizing region has anucleotide sequence that is complementary to an essential nucleic acidsequence of DNA MeTase. Preferably, this region has from about 6 toabout 100 nucleotides. In this embodiment, the oligonucleotide isstabilized, i.e., rendered resistant to exonucleolytic degradation bybase-pairing between the target hybridizing region and theself-complementary region and/or by base-pairing between complementarysequences within the self-complementary region. When the oligonucleotideencounters a DNA MeTase nucleic acid molecule having a complementarynucleic acid sequence, base-pairing between the DNA MeTase hybridizingregion and the self-complementary region of the oligonucleotide isdisrupted and replaced by base-pairing between the DNA MeTasehybridizing region of the oligonucleotide and the complementary nucleicacid sequence of the nucleic acid molecule. This disruption andreplacement of base-pairing takes place because the intermolecularbase-paired structure formed by the hybrid between the target nucleicacid sequence and the target hybridizing region is morethermodynamically stable than the intramolecular base-paired structureformed by this self-complementary oligonucleotide.

A second form of an oligonucleotide according to this embodiment of theinvention operates in a similar way as the first form, but forms adifferent structures upon self-complementary base-pairing. Thisalternatives form forms a hammer-like structure. In this form, theself-complementary region contains oligonucleotide sequences that canbase pair with other oligonucleotide sequences within theself-complementary region. The self-complementary region may alsocontain oligonucleotide sequences that are complementary to thetumorigenicity hybridizing region.

The second significant region of self-stabilized oligonucleotidesaccording to the invention is the self-complementary region. Theself-complementary region contains oligonucleotide sequences that arecomplementary to other oligonucleotide sequences within theoligonucleotide. These other oligonucleotide sequences may be within theDNA MeTase hybridizing region or within the self-complementary region,or they may span both regions. The complementary sequences form basepairs, resulting in the formation of a hairpin structure or ahammer-like structure. Either the hairpin structure or the hammer-likestructure will presumably have loops of 4 or more nucleotides resultingfrom non-base-paired nucleotides. The number of base-pairs to be formedby intramolecular hybridization involving the self-complementary regionmay vary, but should be adequate to maintain a double-stranded structureso that the 3' end is not accessible to endonucleases. Generally, about4 or more base-pairs will be necessary to maintain such adouble-stranded structure. In a preferred embodiment, there are about 10intramolecular base-pairs formed in the self-stabilized oligonucleotide,with the 10 base pairs being consecutive and involving the 3'-mostnucleotides. Of course, the intramolecular base-pairing can be soextensive as to involve every nucleotide of the oligonucleotide.Preferably, this will involve a self-complementary region of about 50nucleotides or less.

Oligonucleotides according to this embodiment may have from 1 to aboutall modified internucleotide linkages, as described for the fourthembodiment. Preferably, at least either the DNA MeTase hybridizingregion or the self-complementary region, and most preferably both, willcontain from about 2 to about all nucleotides being coupled byphosphorothioate and/or phosphorodithioate linkages.

Those skilled in the art will recognize that the features of the variouspreferred embodiments described above can be combined to produceadditional embodiments that may have even greatertumorigenicity-inhibiting activity. Thus, the invention contemplatesmodified tumorigenicity-inhibiting oligonucleotides having everypossible combination of chimeric features, hybrid features, capstructures and self-stabilizing character, all as described herein. Sucholigonucleotides are useful as therapeutic agents for inhibition oftumor growth. For such treatment, oligonucleotides may be administeredintraperitoneally, intranasally, orally or anally. Preferably, sucholigonucleotides will be administered at a concentration of from about 1to about 50 mg/kg body weight.

The following examples are intended to further illustrate certainpreferred embodiments of the invention and are not intended to belimiting in nature.

EXAMPLE 1 Expression of Antisense to the DNA Methyltransferase Gene inY1 Cells Results in Limited DNA Demethylation Cell Culture and DNAMediated Gene Transfer

To directly inhibit DNA methylation in Y1 cells, either the DNA MeTaseantisense expression construct pZαM or a pZEM control vector, Szyf, atal., J. Biol. Chem., 267: 12831-12836 (1992)) was introduced into Y1adrenocortical carcinoma cells by DNA-mediated gene transfer as follows.

Y1 cells were maintained as monolayers in F-10 medium which wassupplemented with 7.25% heat inactivated horse serum and 2.5% heatinactivated fetal calf serum (Immunocorp. Montreal) (Yasumura, et al.,Cancer Res., 26: 529-535 (1988)). All other media and reagents for cellculture were obtained from GIBCO-BRL. Y1 cells (1×10⁶) were plated on a150 mm dish (Nunc) 15 hours before transfection. The pZαM expressionvector (10 μg) was cointroduced into Y1 cells with 1 μg of pUCSVneo as;a selectable marker by DNA mediated gene transfer using the calciumphosphate protocol (Ausubel, et al., 1988, Current Protocols inMolecular Biology, Wiley and Sons, New York). Selection was initiated 48hours after transfection by adding 0.25 mg/ml G418 (GIBCO-BRL) to themedium. For both constructs, G418 resistant cells were isolated and thencloned in selective medium. For analysis of growth in soft agar, 1×10³cells were seeded in triplicate onto 30 mm dishes (Falcon) with 4 ml ofF-10 medium containing 7.5% horse serum, 2.5% FCS, 0.25 mg/ml G418 (fortransfectants) and 0.33% agar solution at 37° C. (Freedman and Shin,Cell, 3: 355-359 (1974)). Cells were fed with 2 ml of medium plus G418every two days. Growth was scored as colonies containing >10⁷ cells, 21days after plating.

EXAMPLE 2 DNA and RNA Analyses

Preparation of genomic DNA and total cellular RNA, labelling (using therandom primer labelling kit from Boehringer Mannheim), blotting RNA onto Hybond-N+(Amersham), and all other standard molecular biologymanipulations were performed according to Ausubel et al., 1988, CurrentProtocols in Molecular Biology. Wiley and Sons, New York. MspI and HpaIlrestriction enzymes (Boehringer Mannheim) were added to DNA at aconcentration of 2.5 units/ug for 8 h at 37° C. Radionucleotides (3000mCi/mmol) were purchased from Amersham.

To confirm that the transfectants bear the introduced construct, DNA wasprepared from this transfectants and subjected to digestion by eitherMspI or HpaII, Southern blot analysis and hydridization with a ³² Plabelled 0.6 kb DNA MeTase cDNA fragment. The results demonstrated thatthe three pZαM transfectants contained significant levels of the DNAMeTase cDNA sequence while the control transfectants were clean.

To test whether the pZαM constructs is expressed in the transfectantsand whether the metallothionein promoter is functional in these cells,the transfectants were cultured with 50 μM of ZnSO₄, RNA prepared atdifferent time points and subsequently subjected to Northern blotanalysis and hybdridization with the ³² P labelled MET 0.6 probe.Transfectants 7 and 9 express substantial amounts of the MET 0.6 cDNA(-1.3 kb chimeric mRNA) even before induction with ZnSO4.

EXAMPLE 3 Demethylation of Specific Genes in Y1 pZαM Transfectants

To verify that expression of pZαM results in demethylation and todetermine whether specific genes were demethylated, HpaII/MspIrestriction enzyme analysis was employed followed by Southern blottingand hybridization with specific gene probes. HpaII cleaves the sequenceCCGG, a subset of the CpG dinucleotide sequences, only when the site isunmethylated while MspI will cleave the same sequence irrespective ofits state of methylation. The pattern of HpaII cleavage of specificgenes in cells expressing pZαM was compared with that of the parental Y1or cells harboring only the vector to determine whether the genes aredemethylated in the antisense transfectants. The state of methylation ofthe steroid 21-hydroxylase gene C21 was analyzed first. (Szyf et al.,Proc. Natl. Acad. Sci. USA, 86: 6853-6857 (1989); Szyf, et al., Mol.Endocrin., 4: 1144-1152 (1990)). This gene is specifically expressed andhypomethylated in the adrenal cortex but is inactivated andhypermethylated in Y1 cells (Szyf et al., Proc. Natl. Acad. Sci. USA,86: 6853-6857 (1989)); Szyf, et al., Mol. Endocrin., 4: 1144-1152(1990)). DNA prepared from Y1, pZαM (Bernards, et al., Proc. Natl. Acad.Sci. USA, 86: 6474-6478 (1989)); Collins et al., J. Exp. Med., 176:1043-1091 (1992)) and pZαM (Bernards, et al., Proc. Natl. Acad. Sci.USA, 86: 6474-6478 (1989)) transfectants was subjected to either MspI orHpaII digestion, Southern blot analysis and hybridization with a 0.36 kbXba-BamHI fragment containing the enhancer and promoter regions of theC21 gene (see Szyf et al., Proc. Natl. Acad. Sci. USA, 86: 6853-6857(1989); Szyf, et al., Mol. Endocrin., 4: 1144-1152 (1990) for physicalmap of the probe). This probe detects 0.36 kb and 0.16 kb HpaIIfragments when the promoter region is fully demethylated (Szyf et al.,Proc. Natl. Acad. Sci. USA, 86: 6853-6857 (1989); Szyf, et al., Mol.Endocrin., 4: 1144-1152 (1990)).

The promoter and enhancer region is heavily methylated in Y1 cells andthe pZEM transfectants. In contrast, the Y1 pZαM transfectants bear apartially demethylated C21 5' region as indicated by the relativediminution of the 3.8 and 2 kb fragments and the appearance of the fullydenothylated faint bands at 0.36 kb as well as the fact that HpaIIcleavage yields partial fragments at 0.56 and -1 kb indicating partialhypomethylation of sites upstream and downstream to the enhancer region.

To determine whether hypouethylation was limited to the enhancer regionor spread throughout the C21 gene locus, similar HpaII digestion andSouthern blot transfer were performed on different preparations of DNAextracted from Y1 cells, a control pZEM (Bernards, et al., Proc. Natl.Acad. Sci. USA, 86: 6474-6478 (1989)) transfectant, and three pZαMantisense transfectants. The filter was hibridized with a 3.8 kb BamHIfragment containing the body of the C21 gene and 3' sequences (Szyf etal., Proc. Natl. Acad. Sci. USA, 86: 6853-6857 (1989); Szyf, et al.,Mol. Endocrin., 4: 1144-1152 (1990) for physical map). Fulldemethylation of this region yields a doublet at ˜1 kb, a 0.8 kbfragment and a 0.4 kb fragment as well as a number of low molecularweight fragments at 0.1-0.2 kb. The C21 locus is heavily methylated inY1 cells as well as the control transfectant as indicated by the highmolecular weight fragments above 23 kb. Only a faint band is present inthe expected 1 kb molecular weight range as well as a partial at 1.9 kbas well as the appearance of new partial fragments in the lowermolecular weight range between 1 and 0.4 kb indicating partialhypomethylation at a large number of HpaII sites contained in the 3'region of the C21 gene (Szyf et al., Proc. Natl. Acad. Sci. USA, 86:6853-6857 (1989); Szyf, et al., Mol. Endocrin., 4: 1144-1152 (1990)).The pattern of demethylation, indicated by the large number of partialHpaII fragments, is compatible with a general partial hypomethylationrather than a specific loss of methylation in a distinct region of theC21 gene.

To determine whether demethylation is limited to genes that arepotentially expressible in Y1 cells such as the adrenal cortex-specificC21 gene (Szyf, et al., Mol. Endocrin., 4: 1144-1152 (1990)) or if thedemethylation is widely spread in the genome, other genes such as themuscle specific MyoD gone as well as the hippocampus specific 5HT1Areceptor gene were analyzed; both genes were hypomethylated.

Another class of genes that might have undergone a specifichypomethylation includes the tumor suppressor genes. The state ofmethylation of two genes from this class was determined, p53 andretinoblastoma (RB) which are both tumor suppressor genes involved incell cycle regulation. Loss of either one of these gene products hasbeen shown to lead to deregulation of the cell cycle and neoplasia(Bernards, et al., Proc. Natl. Acad. Sci. USA, 86: 6474-6478 (1989);Donehoweer, et al., Nature, 356: 215-221 (1992)).

Generation of p53 and retinoblastoma (RB) probes by PCR

Oligoprimers for the 5' region of the mouse p53 gene were selected fromthe published genomic sequence (Accession number: XO1235) (Zakut-Houri,et al., Nature 306: 594-597 (1983)) using the Primer selecting program(PC Gene). The 5' primer corresponding to bases 154-172: 5' TCC GAA TCGGTT TCC ACCC 3' (SEQ ID NO 3) and the 3' primer corresponding to bases472-492 5' GGA GGA TGA GGG CCT GAA TGC 3' (SEQ ID NO 4) were added to aamplification recation mixture containing 100 μg of mouse DNA (fromC2C12 cells) using the incubation conditions recommended by themanufacturer (Amersham Hot tub) (1.5 mM MgCl₂) and the DNA was amplifiedfor 40 cycles of 2 minutes at 95° C., 2 minutes at 55° C. and 0.5minutes at 72° C. The reaction products were separated on a low-meltagarose gel (BRL) and the band corresponding to the expected size wasexcised and extracted according to standard protocols (Ausubel, et al.,1988, Current Protocols in Molecular Biology. Wiley and Sons, New York).

Since the genomic sequence of the mouse RB gene was unavailable throughGenbank we reverse transcribed the retinoblastoma mRNA from 0.5 μg oftotal mouse RNA (from C2C12 cells) using random oligonucleotide primers(Boehringer) with Superscript reverse transcriptase (BRL) underconditions recommended by the manufacturer. The RB sequence wasamplified from the reverse transcribed cDNA using oligonucleotidescorresponding to bases 2-628 of the published cDNA (Bernards et al.,Proc. Natl. Acad. Sci. USA, 86: 6474-6478 (1989)). The oligoprimers usedwere 5' GGA CTG GGG TGA GGA CGG 3' (1-18) (SEQ ID NO 5) and 5' TTT CAGTAG ATA ACG CAC TGC TGG 3' (620-610) (SEQ ID NO 6). The amplificationconditions were as described above.

Using a probe to a 300 bp sequence from the 5' region of the mouse RBcDNA, the level of methylation of this gene was determined in Y1 cellstransfected with a control vector as well as the pZαM transfectants,.Cleavage of this region with HpaII yields 0.6 kb and 0.1 kb fragments.The RB locus is heavily methylated in the control cells as indicated byhybridization of the probe to high molecular weight fragments. Thislocus is partially hypomethylated in the pZαM transfectants as indicatedby the relative diminution in the intensity of the high molecular weightmarkers and the partial presence of the 0.6 and 0.15 kb fragments.

EXAMPLE 4 Nearest Neighbor Analysis

To determine whether expression of antisense RNA to the DNA MeTase geneleads to a general reduction in the level of methylation of the genome,"nearest neighbor" analysis using α-³² P!-dGTP was conducted asdescribed by Razin et al., 1985, in Razin, A., and G. L. Cantoni. (Ed),Biochemistry and Biology of DNA methylation, Allan R. Liss, Inc. N.Y.This assay enables a determination of the percentage of methylatedcytosines residing in the dinucleotide sequence CpG. Transfectants andcontrol DNAs were nicked with DNAaseI, nick translated with a singlenucleotide α-³² P!-dGTP using DNA polymerase I and the labelled DNA wasdigested to 3' mononucleotide phosphates with micrococal nuclease whichcleaves DNA 3' to the introduced α-³² P. The α-³² P! labelled 5αneighbors of dGMP were separated by chromatography on a TLC plate, theresulting spots for dCMP and dC^(met) MP were scraped and counted byliquid scintillation. The results of a triplicate experiment presentedin FIG. 2a (sample autoradiogram) and b (graphic representation) suggestthat a limited but significant reduction in the total level of DNAmethylation (12% for transfectant number 4 and 22% for 7) occurred intransfectants expressing the pZαM construct when compared to the controlline pZEM.

"Nearest Neighbor" analysis was performed as follows: 2 μg of DNA wereincubated at 37° C. for 15 minutes with 0.1 unit of DNAase, 2.5 μl of ³²P-α-dGTP (3000 Ci/mmol from Amersham) and 2 units of Kornberg DNApolymerase (Boehringer) were then added and the reaction was incubatedfor an additional 25 minutes at 30° C. 50 μl of water were added and thenonincorporated nucleotides were removed by spinning through a microconcolumn (Anicon) at maximum speed for 30 seconds. The labelled DNA (20μl) was digested with 70 μg of micrococal nuclease (Pharmacia) in themanufacturer's recommended buffer for 10 hours at 37° C. Equal amountsof radioactivity were loaded on TLC phosphocellulose plates (Merck) andthe 3' mononucleotides were separated by chromatography in one dimension(iso-butyric acid: H₂ O: NH₄ oH in the ratio 66:33:1). The chromatogramswere exposed to XAR film (Eastman-Kodak) and the spots corresponding tocytosine and 5-methylcytosine were scraped and counted in aβ-scintillation counter.

EXAMPLE 5 In Vitro Tumorigenicity Assays

While control Y1 and Y1 pZEM cells exhibit limited contact inhibitionand form multilayer foci, Y1 pZαM transfectants exhibit a more roundedand distinct morphology and grow exclusively in monolayers.

To determine whether the expression of antisense to the DNA MeTaseresults in reversal of the tumorigenic potential, the ability of thetransfectants to grow in an anchorage independent fashion wasdetermined. This assay is considered an indicator of tumorigenicity(Freedman and Shin, Cell 3: 355-359 (1974)). The Y1 pZαM transfectantsdemonstrate an almost complete loss of ability to form colonies in softagar, moreover the colonies that do form contain only a few cells asdemonstrated (FIG. 3B). Growth on soft agar was quantified by visualexamination and presented graphically in FIG. 3. These experimentsdemonstrate that inhibition of DNA methylation by expression of anantisense message to the DNA MeTase leads to loss of tumorigenicity invitro.

EXAMPLE 6 In Vivo Tumorigenicity Assays

Syngenic LAF-1 mice (6-8 week old males) were injected subcutaneously(in the flank area) with 10⁶ cells of each of the Y1 pZαM, Y1 and Y1pZEM transfectants. Mice were monitored for the presence of tumors bydaily palpitation. Mice bearing tumors of greater than 1 cm in diameterwere sacrificed by asphyxiation with CO₂, tumors wore removed bydissection and homogenized in guanidium isothiocyanate. Mice that weretumor free were kept for ninety days and then sacrificed. RNA wasprepared from the tumors by CsCl₂ (Ausubel, et al., 1988, CurrentProtocols in Molecular Biology, Wiley and Sons, New York).

The presence of tumors was determined by palpitation. While all theanimals injected with Y1 cells formed tumors two to three weeks postinjection, the rate of tumor formation in the animals injected with thepZαM transfectants was significantly lower. The results are shown belowin Table I.

                  TABLE I    ______________________________________    Cell line injected                   Tumors  Neovascularization    ______________________________________    Y1             6/6     +++    pZEM 4         5/5     +++    pZαM 4   1/6     ---    PZαM 7   2/6     ---    pZαM 9   2/6     ---    ______________________________________

EXAMPLE 6A In Vivo Inhibition of Tumorigenicity of Human Small LungCarcinoma Cells in a Nude Mouse System

To determine whether inhibition of DNA MeTase by expression of anantisense message results in inhibition of cellular transformation ofhuman carcinomas, a 330 bp sequence containing the translationinitiation site (+155-+481) was amplified using the published human DNAMeTase cDNA sequence using the amplification protocol described above inExample 3 (antisense primer was: 5' GCA AAC AGA ATA AAG AAT C 3' (SEQ IDNO 7), the sense primer was: 5' GTA TGG TGG TTT GCC TGG T 3' (SEQ ID NO8)). The 330 bp sequence was subcloned in the antisense orientation intothe expression vector pZEM as described above for the mouse antisense. Ahuman small lung carcinoma cell line NCI H446 was cotransfected witheither an antisense DNA MeTase expression vector or a control senseexpression vector and a plasmid conferring resistance to hygromycinusing transfection protocols as described above. Hygromycin resistantcolonies were selected and the presence of the transfected antisense wasverified by digestion with EcoRI, Southern blot transfer andhybridization with a 0.4 kb human DNA MeTase cDNA probe. Demethylationof genomic DNA of cells expressing the antisense was verified by nearestneighbor analysis (FIG. 6) as described above and by hybridization withspecific gone probes. The gene encoding the IGF-1 growth factor wasdemethylated in antisense transfectants but not sense controls.

To determine whether the expression of antisense to DNA MeTase resultsin reversal of the tumorigenic potential, the ability of thetransfectants to grow in an anchorage independent fashion was analyzed.The: antisense transfectants lost their ability to form colonies in softagar indicating loss of tumorigenicity in vitro.

Tumor growth in nude mice was evaluated as follows::

4 groups of mice were injected with 10⁶ NCI H446 cells transfected withthe pZαM 5' human MeTase (0.4 kb) antisense expression plasmid and thehygromycin resistance plasmid.

1 group of mice was injected with 10⁶ NCI H446 cells transfected withthe pZαM 5' human MeTase (0.4 kb) sense expression plasmid and thehygromycin resistance plasmid.

1 group of mice was injected with 10⁶ NCI H446 cells bearing thehygromycin resistance plasmid.

1 group of mice was injected with 10⁶ NCI H446 lung cell line.

The mice were followed for in excess of 12 weeks. The results are shownin Table II. These results demonstrate that expression of antisense tothe DNA MeTase inhibited turigenesis In vivo.

                  TABLE II    ______________________________________    TUMOR DEVELOPMENT IN NUDE MICE                                     Latency period                         Number of mice                                     of mice    Transfectant             Number of mice                         developing  developing    clones   injected    tumors      tumors    ______________________________________    pZαM.sup.1 #3             3           0           >12 weeks    pZαM #3             3           0           >12 weeks    pZαM #3             2           0           >12 weeks    pZαM #3             3           0           >12 weeks    pZM.sup.2 #5             3           2           5 weeks    Hyg only.sup.3             3           3           5 weeks    Tumor only.sup.4             3           3           3 weeks    ______________________________________     .sup.1 NCI H446 cells transfected with the pZαM 5' human MeTase (0.     kb) antisense expression plasmid and the hygromycin resistance plasmid     .sup.2 NCI H446 cells transfected with the pZαM 5' human MeTase (0.     kb) sense expression plasmid and the hygromycin resistance plasmid     .sup.3 NCI H446 cells bearing the hygromycin resistance plasmid     .sup.4 NCI H446 lung cell line

Neovascularization

Many lines of evidence suggest that angiogenic potential and metastaticpotential of cell lines area directly related (Liotta, at al., Cell, 64:327-336 (1991)). The tumors that do arise from the pZαM transfectantsexhibit very limited neovascularization while tumors that formed in theanimals that were injected with Y1 cells or control transfectants werehighly vascularized.

RNA from a tumor arising from the Y1pZαM transfectant was isolated andthe level of expression of the 0.6 kb antisense message was comparedwith that observed for the transfectant line in vitro. The isolated RNAswere subjected to Northern blot analysis and hybridization with a ³² Plabelled MET 0.6 fragment. The filter was stripped of its radioactivityand was rehybridized with a ³² P labelled oligonucleotide probe for 18SrRNA as previously described (Szyf et al., Mo. Endocrinol., 4: 1144-1152(1990)). The autoradiograms were scanned and the level of expression ofMET 0.6 was determined relative to the signal obtained with the 18Sprobe. The expression of the antisense message is significantly reducedin the tumors. Thus, it appears that expression of an antisense messageto the DNA MeTase is incompatible with tumorigenesis. Apparently, thesmall number of tumors that did form in animals injected, with the pZαMtransfectants were derived from revertants that lost expression of theantisense to the DNA MeTase under the selective pressure in vivo.

EXAMPLE 7 Relationship of Serum Deprivation and Expression of pZαM in Y1Cells to Apoptotic Death Program

Tumor cells exhibit limited dependence on serum and are usually capableof serum independent growth (Barns and Sato, Cell, 22: 649-655 (1980)).Factors present in the serum are essential for the survival of manynontumorigenic cells. Several lines of evidence have recently suggestedthat the enhanced survivability of tumorigenic cells is associated withinhibition of programmed cell death. For example, the oncogene bcl-2 isnot a stimulator of cell proliferation but rather causes inhibition ofapoptosis (Strasser, et al., Nature, 348: 331-333 (1990)). The tumorsuppressor p53 can induce apoptosis in a human colon tumor derived line(Shaw, et al., Proc. Natl. Acad. Sci., 89: 4495-4499 (1992)) and certainchemotherapeutic agents have been shown to incude apoptosis in cancercells (Collins et al, J. Exp. Med., 176: 1043-1091 (1992)).

Observation of the pZαM transfectants indicated that they exhibitedenhanced dependence on serum and limited survivability under serumdeprived conditions. The effects of serum starvation were studied onpZαM transfectants. pZαM transfectants and control Y1 pZEM transfectants(3×10⁵ per well) were plated in low serum medium (1% horse serum) in sixwell plates, harvested every 24 hours and tested for viability by trypanblue staining (FIG. 6B). While the control cells exhibited almost 100%viability up to 72 hours after transfer into serum deprived medium, theY1pZαM cells showed up to 75% loss of viability at 48 hours (FIG. 6B).

Y1 pZαM cells were plated in starvation medium (1% horse serum) andharvested at 24 hour intervals. Total cellular DNA was isolated from thecells and was subjected to electrophoresis on a 1.5% agarose gelfollowed by transfer to nylon membrane and hybridization with randomlabeled Y1 genomic DNA. After 48 hours in serum starved conditions, pZαMtransfectants exhibit the characteristic 180 bp internucleosomal DNAladder while the control pZEM transfectants show no apoptosis at thistime point.

Y1 pZαM cells were serum starved for 24 hours (2% horse serum),harvested and analyzed by electron microscopy as follows. Cells werefixed in glutaraldehyde (2.5%) in cacodylate buffer (0.1M) for one hourand further fixed in 1% osmium tetroxide. The samples were dehydrated inascending alcohol concentrations and propylene oxide followed byembedding in Epon. Semi-thin sections (1 μM) were cut from blocks withan ultramicrotome, counterstained with uranil acetate and lead citrate.Samples were analyzed using a Philips 410 electron microscope(Maysinger, et al., Neurochem. Intl., 23: 123-129 (1993)).

Electron microscopy of control Y1 pZEM and Y1 pZαM transfectants atvarious magnifications revealed that control cells have a fine uniformnuclear membrane whereas the pZαM cells exhibit the cardinal markers ofapoptosis (Wyllie, et al., Histochem. J., 13: 681-692 (1981))condensation of chromatin and its margination at the nuclear periphery,chromatin condensation, nuclear fragmentation, formation of apoptoticbodies and cellular fragmentation. This set of experiments suggests thatone possible mechanism through whch demethylation can inhibittumorigenesis is by eliminating the inhibition of programmed cell death.

EXAMPLE 8

In this experiment, human small lung carcinoma cells (NCI H446) weretreated with 5 μl lipofectin reagent (Gibco BRL) and oligo (5 μl) in 1ml serum free media for approximately 4 hours (final oligoconcentrations=5 μM). The media was then replaced with 2 ml normalmedium and oligo was added to obtain a concentration of 5 μM. Medium andoligo were then replaced daily for the following 3 days. The oligos usedwere the following:

34: DW2-34B (antisense phosphodiester) 5' CAT CTG CCA TTC CCA CTC TA 3'(SEQ ID NO 9)

35: DW2-35C 5' Phosphorothioate of 34 (SEQ ID NO 10)

36: DW2-36C (random control phosphodiester) 5' CTG ACT GCC AAC TAT GAACA 3' (SEQ ID NO 11)

37: DW2-37D 5' Phosphorothioate of 36 (SEQ ID NO 12)

The cells grew reasonably well, however throughout the growth period,there were less cells in the wells treated with oligo 35 than in theothers and many cells in these wells were floating. Several cells werealso deteched in the wells treated with oligo 37.

Experiment A

In this experiment, cells were grown in presence of the oligos forlonger than in the previous experiment (14 days). The initial treatmentcomprised of 5 μl lipofectin and 10 μl oligo in 1 ml media.Subsequently, media was changed and oligo added (10 μl in 2 ml) dailyfor 9 days and for the final 4 days, to avoid losing cells that werefloating but not necessarily dead, the medium was changed only once andoligo added to the medium every other day.

Cells for this experiment were slow to start growing. During the firstweek of treatment with oligo, cells remained quite sparse and a veryhigh proportion of cells were observed to be round and/or floating.During the second week, as the cells started to grow more nicely, clumpsof cells appeared in the control wells and in the 34 and 36. In thewells treated with oligo 35, there were consistently fewer cells and ahigher proportion of floating cells than in control wells. In addition,the 35 cells that remained attached were more elongated than controls.Similar features were observed to a lesser extent in the 37 cells.Toward the end of the experiment, the control cells seemed moreelongated than they had been previously, though significantly less thanthe 35 cells. Wells 34 and 36 contained more large clusters of cellsthan the others (even controls). On the whole, there were fewer clumpsin wells 35 and 37 than in all the others. Oligo 34 (antisensephosphodiester) appeared to have no effect on cell morphology.

To determine whether treatment with DNA MeTase antisenseoligonucleotides inhibits tumorigenesis in vitro, the ability of thetreated cells to grow in an anchorage independent fashion wasdetermined. Two sets of cells were analyzed: Set A was treated for 15days and Set B was treated for 9 days. The number of cells weredetermined by inspection with the naked eye 18 days after plating. Asshown in FIG. 7, the cells treated with oligo 35 have lost the abilityto grow in an anchorage independent fashion in vitro, indicatinginhibition of tumorigenicity In vitro.

Experiment B

Given the fact that cells did not grow very well in the initial stagesof experiment A, more cells (˜150,000 instead of 80,000) were plated torepeat the experiment. These cells were treated with lipofectin (5 μl)and oligo (10 μl) on day 1 and then the medium was changed and 10 μloligo were added daily for three days and for the next four days, 10 μloligo were added daily and the medium was changed only once.

After the 8 days of treatment, cells in wells 36 and 37 were similar inappearance to the control wells. Only the cells treated with oligo 35looked significantly different from the others in that there had beenless growth and cells appeared on the whole less "clumpy" than controls.The cells treated with oligo 35 again lost their ability to formcolonies in soft agar, indicating reversal of tumorigenicity In vitro.

Dose Curve:

Cells were treated for 5 days with different doses of oligo 35(antisense phosphorothioate): 0.5 μM, 1.5 μM, 5 μM, 15 μH and 50 μN.

    ______________________________________              Initial    Well      lipofectin*                         Initial oligo                                     Daily oligo    ______________________________________    control   5μl     0           0    0.5μM  5μl     1μl(1μM)                                     1μl(0.5μM)    1.5μM  5μl     3μl(3μM)                                     3μl(1.5μM)    5μM    5μl     10μl(10μM)                                     10μl(5μM)    15μM   5μl     30μl(30μM)                                     30μl(15μM)    50μM   5μl     100μl(100μM)                                     100μl(50μM)    ______________________________________     *Lipofectin reagent (Gibco BRL)

Initial treatment with lipofectin and oligo were in 1 ml medium andsubsequently, cells were in 2 ml medium.

Treatment with oligo 35 resulted in dramatic changes in cell morphology.At all doses, formation of larger clusters of cells was inhibited withrespect to the controls. As oligo concentration increased, cells becameless clumpy and more elongated. Increasing numbers of floating cellsappeared, many of which were alive as revealed by viability counts.

Upon treatment with 15 μM oligo, cells became dramatically elongated andno clumps of cells could be seen (see pictures). A high proportion ofcells were floating, however viability was found to be over 50%,suggesting that many of the floating cells are still alive.

EXAMPLE 9 In Vivo Inhibition of Tumorigenicity Using AnitsenseTechnology

In vivo inhibition of DNA methyl transferase expression andtumorigenesis can be achieved by administration of the antisenseoligonucleotides of the present invention to mammals. For example,administration into a mouse can be by slow infusion pump at a rate ofabout 0.5-3.0 nMoles/hr (about 0.15-1.0 mg of an oligonucleotide 20-merper kg of body weight). Alternatively, intravenous injection of about1-5 mg of the oligonucleotide per kg body weight can be made into thetail vein. After about 10 to 21 days the tumors can be excised andanalyzed for DNA methyl transferase expression as well as by observingthe weight and morphology of the tumors. Tumors and DNA methyltransferase levels of mice treated with a control oligonucleotide can becompared.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 12    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (iv) ANTI-SENSE: YES    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    # 20               TCTA    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 24 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (iv) ANTI-SENSE: YES    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    #                24CCAC TCTA    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    - 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#feature              (B) LOCATION: 1..18    #/note= "PRIMER 1-18"NFORMATION:    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    #  18              GG    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 24 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: misc.sub.-- - #feature              (B) LOCATION: 1..24    #/note= "PRIMER 620-610"RMATION:    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    #                24ACTG CTGG    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: misc.sub.-- - #feature              (B) LOCATION: 1..19    #/note= "ANTI SENSE PRIMER"TION:    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    # 19               ATC    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: misc.sub.-- - #feature              (B) LOCATION: 1..19    #/note= "SENSE PRIMER"FORMATION:    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    # 19               GGT    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (iv) ANTI-SENSE: YES    -     (ix) FEATURE:              (A) NAME/KEY: misc.sub.-- - #feature              (B) LOCATION: 1..20    #/note= "Oligo 34: DW2-34BATION:    #phosphodiester)"ntisense    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    # 20               TCTA    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (iv) ANTI-SENSE: YES    -     (ix) FEATURE:              (A) NAME/KEY: misc.sub.-- - #feature              (B) LOCATION: 1..20    #/note= "Oligo 35: DW2-35CATION:    #phosphorothioate)"isense    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    # 20               TCTA    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: misc.sub.-- - #feature              (B) LOCATION: 1..20    #/note= "Oligo 36: DW2-36C (Random                   Control P - #hosphodiester)"    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    # 20               AACA    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: misc.sub.-- - #feature              (B) LOCATION: 1..20    #/note= "Oligo 37: DW2-37D (Random                   Control P - #hosphorothioate)"    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    # 20               AACA    __________________________________________________________________________

What is claimed is:
 1. A method for inhibiting tumor growth comprisingproviding to a cell that expresses a DNA methyltransferase gene aneffective tumor growth-inhibiting amount of an antisense oligonucleotidecomplementary to DNA methyltransferase mRNA.
 2. The method according toclaim 1, wherein the oligonucleotide anneals to a coding sequence of theDNA methyltransferase mRNA.
 3. The method according to claim 1, whereinthe oligonucleotide binds to the start or stop sequence of DNAmethyltranferase mRNA.
 4. The method according to claim 3, wherein theoligonucleotide is self-stabilized.
 5. The method according to claim 1,wherein the oligonucleotide is stabilized by methylphosphonothioateinternucleotide linkages, phosphorothioate internucleotide linkages,methylphosphonate internucleotide linkages, phosphoramidateinternucleotide linkages, a 3' end cap, or a 3' hair-pin loop structure.6. The method according to claim 1, wherein the oligonucleotide is amixed phosphate backbone oligonucleotide having an internal sequencethat activates RNase H and that is flanked on one or both sides bysequences that are unable to activate RNase H.
 7. The method accordingto claim 1, wherein the oligonucleotide has the sequence5'-CATCTGCCATTCCCACTCTA-3' (SEQ ID NO 1).
 8. The method according toclaim 1, wherein the oligonucleotide has the sequence5'-TTGGCATCTGCCATTCCCACTCTA-3' (SEQ ID NO 2).